Producer-cell modification of human immunodeficiency virus type 1: Nef is a virion protein

被引:178
作者
Pandori, MW
Fitch, NJS
Craig, HM
Richman, DD
Spina, CA
Guatelli, JC
机构
[1] UNIV CALIF SAN DIEGO,SCH MED,DEPT MED,LA JOLLA,CA 92093
[2] UNIV CALIF SAN DIEGO,SCH MED,DEPT PATHOL,LA JOLLA,CA 92093
[3] SAN DIEGO VET AFFAIRS MED CTR,LA JOLLA,CA 92093
关键词
D O I
10.1128/JVI.70.7.4283-4290.1996
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Type 1 human immunodeficiency viruses encoding mutated nef reading frames are 10- to 30-fold less infectious than are isogenic viruses in which the nef gene is intact, This defect in infectivity causes nef-negative viruses to grow at an attenuated rate in vitro. To investigate the mechanism of Nef-mediated enhancement of viral growth rate and infectivity, a complementation analysis of nef mutant viruses was performed, To provide Nef in trans upon viral infection, a CEM derivative cell line (designated CLN) that expresses Nef under the control of the viral long terminal repeat was constructed, When nef-negative virus was grown in CLN cells, its growth rate was restored to wild-type levels. However, the output of nef-negative virus during the first 72 h after infection of CLN cells was not restored, suggesting that provision of Nef within the newly infected cell does not enhance the productivity of a nef-negative provirus. The genetically nef-negative virions produced by the CLN cells, however, were restored to wild-type levels of infectivity as measured in a syncytium formation assay in which CD4-expressing HeLa cells were targets, These trans-complemented, genetically nef-negative virions yielded wild-type levels of viral output following a single cycle of replication in primary CD4 T cells as well as in parental CEM cells, To define the determinants for producer cell modification of virions by Nef, the role of myristoylation was investigated, Virus that encodes a myristoylation-negative nef was as impaired in infectivity as was virus encoding a deleted nef gene. Because myristoylation is required for both membrane association of Nef and optimal viral infectivity, the possibility that Nef protein is included in the virion was investigated. Wild-type virions were purified by filtration and exclusion chromatography, A Western blot (immunoblot) of the eluate fractions revealed a correlation between peak Nef signal and peak Levels of p24 antigen, Although virion-associated Nef was detected in part as the 27-kDa full-length protein, the majority of immunoreactive protein was detected as a 20-kDa isoform. nef-negative virus lacked both 27- and 20-kDa immunoreactive species. Production of wild-type virions in the presence of a specific inhibitor of the human immunodeficiency virus type I protease resulted in virions which contained only 27-kDa full-length Nef protein, These data indicate that Nef is a virion protein which is processed by the viral protease into a 20-kDa isoform within the virion particle.
引用
收藏
页码:4283 / 4290
页数:8
相关论文
共 33 条
[21]   LACK OF A NEGATIVE INFLUENCE ON VIRAL GROWTH BY THE NEF GENE OF HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 [J].
KIM, SY ;
IKEUCHI, KJ ;
BYRN, R ;
GROOPMAN, J ;
BALTIMORE, D .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1989, 86 (23) :9544-9548
[22]   CELL-SURFACE CD4 DOWN-REGULATION AND RESISTANCE TO SUPERINFECTION INDUCED BY A DEFECTIVE PROVIRUS OF HIV-1 [J].
LITTLE, SJ ;
RIGGS, NL ;
CHOWERS, MY ;
FITCH, NJS ;
RICHMAN, DD ;
SPINA, CA ;
GUATELLI, JC .
VIROLOGY, 1994, 205 (02) :578-582
[23]   DIFFERENTIAL LOSS OF ENVELOPE GLYCOPROTEIN GP120 FROM VIRIONS OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 ISOLATES - EFFECTS ON INFECTIVITY AND NEUTRALIZATION [J].
MCKEATING, JA ;
MCKNIGHT, A ;
MOORE, JP .
JOURNAL OF VIROLOGY, 1991, 65 (02) :852-860
[24]   EXPRESSION OF THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 (HIV-1) NEF GENE DURING HIV-1 PRODUCTION INCREASES PROGENY PARTICLE INFECTIVITY INDEPENDENTLY OF GP160 OR VIRAL ENTRY [J].
MILLER, MD ;
WARMERDAM, MT ;
PAGE, KA ;
FEINBERG, MB ;
GREENE, WC .
JOURNAL OF VIROLOGY, 1995, 69 (01) :579-584
[25]   THE HUMAN IMMUNODEFICIENCY VIRUS-1 NEF GENE-PRODUCT - A POSITIVE FACTOR FOR VIRAL-INFECTION AND REPLICATION IN PRIMARY LYMPHOCYTES AND MACROPHAGES [J].
MILLER, MD ;
WARMERDAM, MT ;
GASTON, I ;
GREENE, WC ;
FEINBERG, MB .
JOURNAL OF EXPERIMENTAL MEDICINE, 1994, 179 (01) :101-113
[26]   REINFECTION RESULTS IN ACCUMULATION OF UNINTEGRATED VIRAL-DNA IN CYTOPATHIC AND PERSISTENT HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INFECTION OF CEM CELLS [J].
PAUZA, CD ;
GALINDO, JE ;
RICHMAN, DD .
JOURNAL OF EXPERIMENTAL MEDICINE, 1990, 172 (04) :1035-1042
[27]  
RHEE SS, 1994, J IMMUNOL, V152, P5128
[28]   PROLINE-RICH (PXXP) MOTIFS IN HIV-1 NEF BIND TO SH3 DOMAINS OF A SUBSET OF SRC KINASES AND ARE REQUIRED FOR THE ENHANCED GROWTH OF NEF(+) VIRUSES BUT NOT FOR DOWN-REGULATION OF CD4 [J].
SAKSELA, K ;
CHENG, GH ;
BALTIMORE, D .
EMBO JOURNAL, 1995, 14 (03) :484-491
[29]   A CONSERVED DOMAIN AND MEMBRANE TARGETING OF NEF FROM HIV AND SIV ARE REQUIRED FOR ASSOCIATION WITH A CELLULAR SERINE KINASE-ACTIVITY [J].
SAWAI, ET ;
BAUR, AS ;
PETERLIN, BM ;
LEVY, JA ;
CHENGMAYER, C .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (25) :15307-15314
[30]   HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NEF INCREASES THE EFFICIENCY OF REVERSE TRANSCRIPTION IN THE INFECTED CELL [J].
SCHWARTZ, O ;
MARECHAL, V ;
DANOS, O ;
HEARD, JM .
JOURNAL OF VIROLOGY, 1995, 69 (07) :4053-4059